This invention relates broadly to the field of medical biology and more specifically to:
(a) Endocrinology, physiology and clinical medicine PA1 (b) Microbiology and cell transformation PA1 (c) Human cell and microbial cell fermentation systems. PA1 (1) Transformation of rate of growth, morphology and structure PA1 (2) Transformation of function PA1 (3) Inter-combination of (1) and (2).
Since the time of the discovery of the hormone insulin and its use in the treatment of humans with diabetes mellitus, insulin has been obtained and prepared commercially from bovine and porcine pancreas. Human insulin from human pancreas has been achieved only on a laboratory scale. Insulins from a host of animal species ranging from fish to elephants and whales have been isolated and studied on a laboratory scale. In recent years, the shortage of bovine and porcine pancreas has necessitated a search for insulin production from other sources or by other processes. Moreover, although bovine and porcine insulins have been used for several years, disadvantages exist due to the species and immunologic differences which are known to incite anti-insulin antibody formation in man following prolonged use. Hence, methods for achieving production of human insulin, which is highly advantageous both in terms of bioactivity and non-antigenicity, have been under study by several scientists.
Two approaches have been tried to produce human insulin. These are: (1) chemical synthesis of human insulin, and (2) the production of human insulin by cultivation of human insulin producing beta cells by in vitro tissue culture systems. Chemical synthesis of human insulin on a large scale has not as yet been achieved due to the complexity of the chemical steps involved in the synthesis as well as the multifold cost of such synthesis procedures as compared with the present cost of producing bovine-porcine insulin. In regard to tissue culture, all methods reported thus far involve primary cultures of small explants or primary monolayers of insulin producing pancreatic beta cells obtained from a host of animal species as well as man. However, such primary cultures do not survive long and cannot be propagated serially to achieve a sufficient number of beta cells in order to produce insulin on a large scale.
The present inventors have also discovered a new technique of growing both animal and human beta epithelioid cells producing insulin in serial secondary cultures, as described, by which one can achieve reasonable amounts of human or animal insulin. This new technique, although far superior to other tissue culture methods described by others, is at present not sufficient to obtain insulin or human insulin on a large scale since the rate of proliferation of human beta cells in vitro culture is relatively limited. Hence, the present invention includes a new process wherein one transforms a rapidly proliferating cell system with genetic information obtained from human insulin producing cells to produce human insulin on a large scale.
In the field of microbiology, several experiments have been performed over the past few years to transform cells. These include:
(1) Transformation of rate of growth and morphology and structure has been achieved using physical agents such as X-rays, alpha, beta and gamma ray irradiations; by chemical methods using chemical mitogens both organic and inorganic and microbial metabolites such as those belonging to the Actinomycin group; and by biological means by placing cells in foreign environments or growing them in contact with other types of cells to achieve an inter-cell communication response. Examples of these range from the transformation of specialized ectodermal cells to keratinized and even fibroblastic cells when placed in exposed or hostile environments, to the transformation of bacteria to resistant strains when placed in prolonged or chronic contact with small sublethal doses of the respective antibiotics.
(2) Transformation of function or part of the functions or functional characteristics has been achieved with the bacteria E. coli. E. coli that were susceptible to one antibiotic were transformed into a resistant strain by transfer of the genetic material (DNA) from another E. coli strain that was already resistant to the particular antibiotic. This has been achieved by extracting plasmid, DNA, from the resistant bacteria and transferring it to a non-resistant strain by using a virus as a carrier. This has been extended by serial transfers to obtain a E. coli strain resistant to several antibiotics by transforming a susceptible non-resistant strain with genetic material (DNA) from different E. coli resistant to different antibiotics.
Finally, E. coli has been transformed to produce a single protein sequence contained in the toad bladder by transferring genetic material (DNA) from cells from the toad bladder.
In all these above cited instances, purified or native DNA has been used as the gene-information carrying the transforming principle and a virus has been employed to carry the genetic information from the parent transforming cell to the recipient transformed cell. Hence, it is possible that besides the appearance of the functional characteristics of the donor E. coli or toad bladder cell in the recipient transformed cell, there may also appear the functional characteristics in terms of protein sequences of the carrier virus which also becomes incorporated into the genetic sequence of the transformed cell.
Biotransformation in antibiotic producing cultures has been successfully achieved by one of the inventors. Streptomyces aurofaciens which produces chlortetracycline was biotransformed, using the functional genome from Streptomyces pimprina which produces the anti-fungal antibiotic thiolutin. The biotransformed Streptomyces aureofaciens in addition to producing chlortetracycline, also produced thiolutin.
The inventors have also transformed functionally non-specific human squamous cells from the buccal (oral) cavity with the functional genome from human insulin producing cells to make the buccal cells produce insulin, as measured by specific radioimmune assay.
For transforming a rapidly proliferating cell system such as microorganisms with the genetic materials from human insulin producing cells, the bacterium E. coli has been studied by several workers. However, this has not been successful so far. E. coli which is a prokaryotic cell (without a definitive nucleus) would appear to be a poor model to attempt transformation with genetic materials from evolved eukaryotic cells (with a definite nucleus), since the E. coli would not have the appropriate nuclear network to incorporate genetic segments from human cells concerned with insulin production. Also, E. coli is not known to produce any long chain amino acids which would be essential to synthesize insulin. However, fungi which proliferate rapidly are eukaryotic cells and are known to produce long chain amino acid sequences as some antibiotics. Hence, we have used a primitive fungus for transformation with the functional genome from human insulin producing cells to produce insulin.